Microbial Control Microbiology Laboratory - Mehtods Used to Kill Microbes

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From the Virtual Microbiology Classroom on
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Image Compound microscope objectives, T. Port
2

• Laboratory Exercise 4
• Microbial Control
• Physical, Chemical
• Chemotherapeutic

Image Zones of bacterial inhibition from
antibiotic disks, T. Port
From the Virtual Microbiology Classroom on
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3
What am I going to learn from Lab Topic
4?Microbial Control

• Investigate the effectiveness various agents of
  control.
• Assess the effectiveness of heat, and of chemical
  disinfectants, in killing vegetative cells versus
  endospores.
• Evaluate ultraviolet radiation as a mechanism of
  control.
• Examine the fundamentals of antibiotic
  sensitivity testing.

Please plug in your microincinerators.
Image Chimp brain in a jar, Gaetan Lee
From the Virtual Microbiology Classroom on
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Action of Antimicrobial Agents

• Many types of chemical and physical microbial
  controls
• Modes of action fall into two basic categories
• 1. Alteration of bacterial cell walls or
  cytoplasmic membranes
• 2. Interference with protein and nucleic acid
  structure

You will be using 4 TSY plates in todays lab.
Lets pour them now.
Image Antibiotic Modes of Action, J. Raghu
From the Virtual Microbiology Classroom on
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1. Alteration of Cell Walls Membranes

• Cell wall maintains integrity of cell.
• When disrupted, cannot prevent cell from bursting
  due to osmotic effects.
• Cytoplasmic membrane contains cytoplasm and
  controls passage of chemicals into and out of
  cell.
• When damaged, cellular contents leak out.
• Viral envelope responsible for attachment of
  virus to target cell.
• Damage to envelope interrupts viral replication.
• Nonenveloped viruses have greater tolerance of
  harsh conditions.

Image Viral life cycle, National Academy of
Sciences
From the Virtual Microbiology Classroom on
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2. Interference with Proteins Nucleic Acid
Structure

• Protein function depends on 3-D shape.
• Extreme heat or certain chemicals denature
  proteins.
• Nucleic Acids can be damaged or destroyed by
  chemicals, radiation, and heat.
• Can produce fatal mutations.
• Can halt protein synthesis through action on RNA.

From the Virtual Microbiology Classroom on
ScienceProfOnline.com
Images Levels of protein structure, M Ruiz, DNA,
Richard Wheeler
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Relative Susceptibility of Microorganisms
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Protocol for Testing a Control Agent

• The basic principle for testing any control
  agent, (whether temperature, chemical or
  antibiotics) is always the same
• 1. Expose the organism to the agent.
• 2. Remove the agent.
• 3. Put organisms in favorable growth medium.
  
• 4. Look for reproduction of organisms.

From the Virtual Microbiology Classroom on
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Methods of Microbial Control That We Will Be
Examining

• Physical
• Heat
• UV
• Chemotherapeutic
• Antimicrobic drugs, like antibiotics
• Chemical
• Bleach, other chemical disinfectants

From the Virtual Microbiology Classroom on
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• Effects of High Temperatures
• Denaturation of proteins
• Interference with integrity of cytoplasmic
  membrane and cell walls
• Disruption of structure and function of nucleic
  acids
• Boiling
• - Kills vegetative cells of bacteria, protozoa
  and fungi,
• and most viruses in 10 minutes (at sea
  level).
• - Boiling time is critical.
• - Some heat is lost as steam.
• - Endospores, protozoan cysts, and some viruses
  can
• survive boiling
• Autoclaving
• - Pressure applied to boiling water prevents
  steam from
• escaping.
• - Boiling temperature increases as pressure
  increases.

Image Autoclave, Astell Scientific
From the Virtual Microbiology Classroom on
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A Microbial Control Using Heat
40
60
80
100

• Each pair of lab partners should inoculate 4
  tubes of nutrient broth with E. coli and 4 tubes
  with Bacillus species (spp.).
• Label the tubes clearly indicating which microbe
  they contain and include your initials.
• You will be exposing each tube of E. coli and
  each tube of Bacillus to different temperatures.
• Label each of the 4 E. coli tubes with the
  temperature it will be exposed to 40oC, 60oC,
  80oC, and 100oC.
• Label each of the 4 Bacillus tubes with the
  temperature it will be exposed to 40oC, 60oC,
  80oC, and 100oC.
• Place each tube into the appropriate water bath
  for 10 minutes.
• Remove the tubes after 10 minutes at temperature.
  Put them into your SAVE test tube rack.

From the Virtual Microbiology Classroom on
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How to Inoculate a Broth Medium
Image Microincinerator, T. Port Test tubes,
Source unknown
From the Virtual Microbiology Classroom on
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13
Methods of Microbial Control That We Will Be
Examining

• Physical
• Heat
• Ultra Violet Radiation
• Sunlight contains the complete spectrum of short
  to long wavelengths of light.
• The short, invisible ultraviolet (UV) wavelengths
  are injurious to non-photosynthetic bacteria.
• UV radiation is strongly absorbed by proteins and
  nucleic acids.
• UV radiation may cause enzyme inactivation,
  genetic mutation or death.
• Chemotherapeutic
• Chemical

Image Sun's corona as seen in deep ultraviolet
by the Extreme ultraviolet Imaging Telescope, NASA
From the Virtual Microbiology Classroom on
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B. Effectiveness of UV Radiation as a Way to
Control Bacteria

• Use 2 TSY Plates.
• Inoculate each by dipping a sterile swab in the
  culture tube of E coli and then wiping the swab
  over the entire surface of the agar.
• Expose both UV radiation for 5 minutes in the
  UV box.

With lid
Without lid
From the Virtual Microbiology Classroom on
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15
Methods of Microbial Control That We Will Be
Examining

• Physical
• Heat
• Ultra Violet Radiation
• Chemotherapeutic
• Antimicrobic drugs, like antibiotics
• Chemical

From the Virtual Microbiology Classroom on
ScienceProfOnline.com
16
C. Effect of Antimicrobials

• Antimicrobics are drugs used in the
• treatment of infectious disease.
• Sensitivity disks can show us
• which antimicrobic will be most effective in
  
• controlling an organism.
• The disks are impregnated with the antibiotic.
• A nutrient agar plate is uniformly inoculated
  with bacteria and the disks are placed on the
  media.
• You will paint one TSY plate with Staph epi and
  one with E. coli, and then expose both dishes to
  the same types of antibiotics.
• Q Why are we using Staph vs. E. coli?
• Over the incubation period, the antimicrobial
  diffuses in all directions out from the disk.
• If the microbe is sensitive to the specific
  antimicrobial in question, a zone of inhibition
  (an area without bacterial growth) will occur
  around the antibiotic.

Image Zones of bacterial inhibition from
antibiotic disks, T. Port
From the Virtual Microbiology Classroom on
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17
Methods of Microbial Control That We Will Be
Examining

• Physical
• Heat
• UV
• Chemotherapeutic
• Antimicrobic drugs, like antibiotics
• Chemical
• Bleach other chemical disinfectants

From the Virtual Microbiology Classroom on
ScienceProfOnline.com
18
Chemical Microbial Control

• Chlorine vs. Lysol
• The purpose of this part of lab is to compare the
  effectiveness of chlorine and Lysol in killing
  bacterial vegetative cells and destroying
  endospores.
• We will compare both the agent and the strength .
  

Image Lysol on shelf, Bnilsen
From the Virtual Microbiology Classroom on
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• Chlorine Mode of Action
• Used to disinfect water and for cleaning surfaces
  (e.g. floors, counters) and has proven effective
  in destroying HIV.
• Kills microbes by inhibiting enzyme activity and
  oxidizing cellular contents so that they no
  longer perform normal metabolic functions.
• Chlorine reacts with organic materials in the
  cell and is used up.
• To be effective, chlorine concentrations must be
  high enough to allow chlorine to attach to all
  the organic material present and still have some
  residual.
• Lysol (Phenol) Mode of Action
• First used by Lister in the mid 1800's to
  sterilize surgical instruments (aka carbolic
  acid).
• Phenols exert their germicidal effect by
  denaturing proteins and destroying the selective
  permeability of the cell membrane (which makes
  cells leak).

Image Lysol on shelf, Bnilsen
From the Virtual Microbiology Classroom on
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D. Chemical Microbial Control

• 1. Materials
• a. The chemical agent and type of bacteria that
  you were assigned.
• b. 10 tubes of broth
• c. 10 blank (empty) tubes
• 2. Label 1 blank and 1 broth tubes with dilution
  type of bacteria
• a. label strength (LS) one blank and two broth
  tube
• b. 10-1 one blank and one broth tube
• c. 10-2 one blank and one broth tube
• d. 10-3 one blank and one broth tube
• e. 10-4 one blank and one broth tube
• Working with the blank tubes, pipet 10 ml of each
  of the agent dilutions (i.e. label strength,
  10-1, 10-2) into the appropriately labeled empty
  tube.

Set up for E. coli and Bacillus
LS
10-1
10-2
10-3
10-4
10 min
5 Broths
LS
10-1
10-2
10-3
10-4
Original chemical solution inoculation
5 Blanks
From the Virtual Microbiology Classroom on
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• Confused?
• Here are links to fun resources that further
  explain
• microbial control
• Microbial Control Laboratory Main Page on the
  Virtual Microbiology Classroom of Science Prof
  Online.
• Control of Microbial Growth, Todars Online
  Textbook of Bacteriology.
• Play Pandemic 2 a video game of strategy, where
  you try to become a successful pandemic microbe
  and infect the world. My 14-year old, crazy-smart
  daughter recommends this one to you.
• Play Disease Defenders educational video game,
  Rice University.
• Pasteurs Experiment in which he used heat to
  control microbes animation and quiz from WH
  Freeman.

Smart Links
(You must be in PPT slideshow view to click on
links.)
From the Virtual Microbiology Classroom on
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Are microbes intimidating you?Do
yourself a favor. Use the
Virtual Microbiology
Classroom (VMC) !The VMC is full of resources
to help you succeed, including

• practice test questions
• review questions
• study guides and learning objectives

You can access the VMC by going to the Science
Prof Online website www.ScienceProfOnline.com
Images E. coli, Giant Microbes Prokaryotic
cell, Mariana Ruiz